Protocol for PPP1R15A-inhibited mouse model establishment with subcutaneous B16F1 tumor and single-cell analysis

Summary Here, we present a protocol for exploring the effects of PPP1R15A inhibitor, Sephin1, on antitumor immunity of B16F1 subcutaneous tumor in mice. We describe steps for constructing single-cell transcriptome and TCR libraries, sequencing, and using sequencing data for the integration of expression and TCR data. We then detail procedures for gene differentiation, regulon and cell-cell communication analysis, and validation of single-cell analysis results. For complete details on the use and execution of this protocol, please refer to Wang et al.1


Highlights
This procedure describes the construction of mouse model, including the chemical injection and tumor cell implantation.Mice used are six-to eight-week-old male C57BL/6 mice.
1. Separate the C57BL/6 mice into two groups randomly: the control group and the Sephin1 group.Each group should contain about 8-12 mice.2. Chemical injection.
a. Inject the Sephin1 group mice with 100 mL Sephin1 solution intraperitoneally, while the control group with equal volume of control solution.b.Inject both groups three times per week for two weeks (Day 1, 3, 5 in each week).3. Tumor implantation.Proceed with tumor implantation two days after the last Sephin 1 pre-treatment.a. Collect cultured B16F1 cells with trypsinization as described above.b.Resuspend the cells with PBS at a concentration of 2:4 3 10 6 /mL.Then measure cell concentration and viability with AO/PI using the equipment Cellometer K2. c. Inject each mouse with 125 mL of cell suspension subcutaneously at the left side of mouse abdomen for a total of 3310 5 living cells per mouse.d.Measure the tumors are measured every 2-3 days for a total of 15 days post-injection.
i. Measure the shortest and longest tumor diameters (d and D) with a Vernier caliper every one or two days.ii.Calculate the tumor volume (V) as: V = d 2 3 D=2.
CRITICAL: Keep the prepared B16F1 cells at 4 C before implantation implant the cells within 30 min after preparation.

Timing: 4 h
This procedure describes the single-cell sample preparation process, including the separation of mouse peripheral blood mononuclear cells (PBMCs) and tumor immune cells.c.Measure the total volume of blood sample, EDTA and PBS.Then add the mixture onto the surface of an equal volume of Ficoll-Paque PREMIUM in 15 mL centrifuge tube.d.Centrifuge at 18 C-20 C with 400 3 g for 20 min with an acceleration of 5 and a deceleration of 3. e. Carefully remove the tubes from the centrifuge to avoid disturbing the layers.PBMCs are enriched at the interphase fluffy white layer (Figure 5).Then collect the mononuclear cells carefully without pipetting the adjacent plasma and Ficoll-Paque PREMIUM fluids.
Note: If needed, remove the red blood cells by resuspending the cells in ACK lysing buffer and incubating for 3 min at room temperature.Add 1-2 mL of ACK lysing buffer into each sample.After three minutes, add 10 mL PBS into each tube.Then centrifuge the cells with 400 3 g, 4 C for 5-10 min and wash cells once with PBS.
f. Stain the cells with AO/PI and quantify the cells using the machine Cellometer K2 (Nexcelom Bioscience).

Note:
The hemocytometer and trypan blue can also be used for cell calculation as an alternative.The percentage of living cells should be more than 70% and the number of living cells should be larger than 2310 5 to ensure experimental success.Note: All the gene expression libraries are sequenced with a data size of 270 millions of clusters.The data size of each TCR-enriched library is 34 millions of clusters.In addition, the average number of reads per cell is not less than 30 M in the expression data and not less than 15 M in the TCR-enriched data.
Note: This step is performed on R.
i. Analyze and integrate the TCR contig matrix using scRepertoire (version 1.2.1). 2 ii.Integrate with the gene expression data.
iii.Separate and annotate TCRs by their distribution in one sample.
Note: If the percentage of the clone number of one clonotype in all clones of the sample was between 0.1 and 1, the clonotype was classified as ''hyperexpanded''; if the percentage was between 0.01 and 0.1, the classification was ''large''.''Medium'' was used to denote a percentage between 0.001 and 0.01, and ''small'' indicated a percentage between 10 À4 and 0.001.
c. Integration of expression data and TCR enrichment data.
Note: This step is performed on Python and R. Plots of different TCR types are made with ggplot2 (version 3.3.5).
i. First, calculate the TCR clonotype frequency by scRepertoire and save the results to csv file.This step is completed by R.

EXPECTED OUTCOMES
This protocol provides a detailed protocol including mouse model construction, cell isolation and single-cell data analysis.After 14-15 days tumor growth, the tumor volume in the control group is around 200-300 mm 3 , and in the Sephin1 treated group is around 600 mm 3 (Figure 1).The average tumor weight at that time point in the control group is around 0.2 g, and around 0.5 g in the Sephin1 group (Figure 2).About 10 6 PBMCs can be collected from 100-200 ml of blood sample.The tumor immune cells are collected through FACS sorting, and live CD45+ cells make up about 1-2% of cells of dissociated tumor.The main immune cell types from all samples include B cells, T cells, NK cells, NKT cells, macrophages, and various other cell types (Figure 3).In addition, the total number of immune cells in each mouse spleen is about 5 3 10 7 , and about 10% CD8+ T cells can be isolated by the MojoSort Mouse CD8 T Cell Isolation Kit.

QUANTIFICATION AND STATISTICAL ANALYSIS
All statistical analyses were conducted using GraphPad Prism 7 (La Jolla, CA, USA) or R (version 3.6.3).Statistic differences between groups were calculated with different methods depended on the data characteristics, including unpaired two-tailed Student's t-test, Chi square test, or Wilcoxon

LIMITATIONS
In the mouse model construction process, this protocol concentrates on the subcutaneous tumor construction from the melanoma cell line B16F1.Other tumor types and tumor model construction methods are not used in this protocol, which limits the application scope of this protocol.
In the single-cell library construction procedure, only the expression library and TCR enrichment library are constructed.The BCR enrichment library may also be useful and have important functions in the antitumor immune process.
In addition, this protocol only explores the composition of immune cells in the tumor microenvironment and peripheral blood.Immune cells in the lymph glands and spleens can also play a key role in the antitumor immunity.Change in the tumor composition over time is also important to explore the function of Sephin1, but not fully studied in this research.

TROUBLESHOOTING Problem 1
When making the Sephin1 solution, the powder cannot be fully dissolved in the solvent.

Protocol Potential solution
When dissolving the Sephin1 powder, first, dissolve the powder in the proper amount of DMSO, and then diluting the Sephin1-DMSO into the proper amount of PBS.After that, incubate the solution in 37 C to maximize the solubility.The solution needs to be mixed thoroughly every time before use.

Problem 2
In the tumor sample, there is a low fraction of living immune cells, which makes it difficult for their isolation.

Potential solution
The mouse should be killed immediately before the tumor tissue collection to shorten the wait time.When digesting the single-cell suspension from tumor tissue, make sure that the standard dissociation protocol from the dissociation kit is carried out exactly as the protocol described.After dissociation, the cell suspension should be filtered by 100 mm cell strainer firstly to remove aggregated cells.

Problem 3
The expression data is large, and the data process can be time consuming with large memory requirements.

Potential solution
Use the server with multithread and large RAM.The server with threads more than 32 and RAM larger than 500 GB is preferred.Better performance of the server can shorten the running time of reads mapping and data integration process.

Problem 4
Some samples may have erythrocyte contamination and influence the expression results, especially in PBMC samples.

Potential solution
The best way is to remove the red blood cells as thoughly as possible during the sample preparation process.If there are still remaining red blood cells(RBCs) during single-cell data analysis, several steps should be done before analysis.First, after the integration step, expression data should be scaled by adding the vars.to.regress parameter(mouse.data<-ScaleData(mouse.data,vars.to.regress = "percent.mt"))to minimize the influence of RBCs-related genes.Second, clusters identified as red blood cells should be removed from the data.The remaining cells are used for further analysis.

Problem 5
There might be some conflicts between the operation system and different versions of R package, resulting in the installation failure of some packages.

5 .
Mouse tumor immune cell separation.Collect the tumors from mice 15 days post-injection.Choose two mice for tumor immune cells separation in each group.Measure the tumor weight before the immune cell separation procedure.a. Collect the tumor tissues and cut them into small pieces (approximately 1-2 mm 3 ).b. Digest the tumor tissues with the mouse tumor dissociation kit (Miltenyi/MACS, 130-096-730), and then filter the prepared cells with the 30 mm MACS SmarterStrainer.c. Incubate the cells with mouse CD45-specific antibody (BioLegend, 157607) for 30 min and then incubate with propidium iodide solution with the volume ratio of 1:100 immediately before FACS cell sorting (Nexcelom Bioscience, CS1-0109-5mL).d.Sort the cells on a BD SORP FACSAria with standard procedure to capture CD45-positive and living cells.6. Use the isolated single-cell suspensions for 103 library construction following standard procedures.The library construction kits include the Chromium Next GEM Single Cell 5 0 Library & Gel Bead Kit v1.1 (16 rxns, PN-1000165), Chromium Single Cell 5 0 Library Construction Kit (16 rxns, PN-1000020), Chromium Single Cell V(D)J Enrichment Kit (Mouse T cell, 96 rxns, PN-1000071), Chromium Next GEM Chip G Single Cell Kit (48 rxns, PN-1000120) and Single Index Kit T Set A (96 rxns, PN-1000213).7. Sequence all samples on an Illumina NovaSeq PE150 platform.

Figure 1 .
Figure 1.Tumor growth curve in the normal and Sephin1 group 1 Data are represented as mean G SEM.

Figure 2 .
Figure 2. Tumor weight in the normal and Sephin1 group 1 Data are represented as mean G SEM.

Figure 3 .
Figure 3. Cell type composition in different samples 1
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NotePrepare in advance.Storage at À20 C for less than two weeks and À80 C for two weeks -6 months.STAR Protocols 4, 102616, December 15, 2023 Protocoliii.Third, mapping the TCR type results to the Seurat object containing the expression data, and make the DimPlot graphs and calculate the TCR type specific genes.